Without limiting the scope of the invention, its background is described in connection with the development of lethal toxins such as botulinum toxin into useful pharmaceuticals. The disease term “botulism” was derived from the Latin word for sausage, botulus, on the basis of a fatal outbreak in Germany in the 1800s caused by partially cooked sausage. Botulism was later determined to be due to toxins elaborated by the Clostridium botulinum organism, originally isolated in 1895. C. botulinum is a gram-positive, spore-forming, anaerobic rod commonly found on plants, in soil, water, and the intestinal tracts of animals. Botulinum toxin was first purified in 1928 and is among the most toxic substances known. Botulinum toxins act to cause paralysis at four different sites in the body: the neuromuscular junction, autonomic ganglia, postganglionic parasympathetic nerve endings, and postganglionic sympathetic nerve endings that release acetylcholine (Ach).
Several different strains of Clostridium, including C. butyricum, C. baratii, and C. argentinense, have been identified that produce antigenically distinct forms of pharmacologically similar botulinum neurotoxins (abbreviated either as BTX or BoNT). Eight major serotypes of botulinum neurotoxins elaborated by bacteria of the Clostridium genus are now known: A, B, C (C1 and C2), D, E, F, and G. Subtypes of the serotypes may be closely related or more distant. For example BoNT-A1 and A2 are 95% homologous while BoNT-A3 and A4 are respectively 81% and 88% homologous to BoNT-A1. Botulinum toxin type A (BoNT-A) is the most potent toxin, followed by the B and F toxin types. It is these types that have been exploited commercially to date.
BoNT-A (Oculinum, now BOTOX® brand, Allergan, Inc.) was FDA approved in 1989 for medical treatment of blepharospasm (uncontrolled blinking), strabismus (crossed eyes), Meige's syndrome (bilateral blepharospasm with concurrent dystonia of the lower face), and hemifacial spasm. Approval to treat cervical dystonia, a neurological movement disorder causing severe neck and shoulder contractions was received in December 2000. BOTOX® was approved in the UK for axillary hyperhidrosis (excessive sweating) in 2001 and, in the same year, BOTOX® was approved in Canada for axillary hyperhidrosis, focal muscle spasticity, and cosmetic treatment of wrinkles at the brow line. In 2002, the U.S. FDA announced the approval of BOTOX® Cosmetic to temporarily improve the appearance of moderate-to-severe frown lines between the eyebrows (glabellar lines). In July 2004, the FDA approved BOTOX® to treat primary axillary hyperhidrosis.
BoNT-B has also been developed for clinical use and several products are currently commercially available (e.g., MyoBloc® in the United States and NeuroBloc® in Europe, Solstice Neurosciences). MyoBloc® was FDA approved in 2000 for treatment of cervical dystonia. BOTOX® BoNT-A is considered to be 50-100 times more potent than Myobloc® BoNT-B for any given specific treatment.
A number of different manufacturers worldwide are now producing purified BoNT-A under various brand names including Xeomin® (Merz Pharma, Germany), Prosigne® (Lanzhou Inst. For Biol. Prod., China), and Neuronox® (Meditoxin® in Korea, Medy-Tox, Inc., Korea). As with other biological products, the biological activity for new batches must be determined. This is particularly critical for drugs such as botulinum toxin, which have considerable toxicity and for which restoration of normal neuromusclar activity is prolonged. Botulinum toxin delivered intramuscularly acts at the neuromuscular junction to cause muscle paralysis by inhibiting the release of Ach from presynaptic motor neurons. The peak of the paralytic effect occurs 4-7 days after injection. Approximately 2 months after the administration of botulinum toxin, the axon begins to expand, and new nerve terminal sprouts emerge and extend towards the muscle surface. These new nerve sprouts re-establish the motor nerve unit and muscle paralysis is reversed typically within 2-4 months. Overdose is sufficiently critical and recovery sufficiently prolonged that an antivenin is available in the case of accidental overdose.
Doses of all commercially available botulinum toxins are expressed in terms of units of biologic activity. One unit of botulinum toxin corresponds to the calculated median intraperitoneal lethal dose (LD50) in mice. See Hoffman R O, Helveston E M. “Botulinum in the treatment of adult motility disorders” Int Ophthalmol Clin 26 (1986) 241-50. Although certainly not ideal, the LD50 assay has been retained due to its high sensitivity. The definition of unit applies to all forms of commercially available toxins, in spite of the fact that a standardized approach to potency testing has not been adopted and inter-manufacturer differences in mouse LD50 assay protocols have led to considerable variability in the per unit activity for different products. For example, the bio-equivalence ratio of the Dysport® brand of BoNT-A (Ipsen Pharmaceuticals, France) to Botox® has been suggested to be between 2.5:1 and 4:1 by various studies in patients with blepharospasm and hemifacial spasm. As a consequence, when communicating recommended botulinum toxin dosage in particular indications, it is has been considered important to specify the particular brand of toxin being used because the units used in labeling are product specific and non-interchangeable. Further complications arise due the fact that BoNT is a large and relatively labile molecule. Proper reconstitution of the product is important and shelf life is quite limited.
Botulinum toxin is capable of inducing formation of antibodies in humans leading to decreased effect of the toxin over time. See Frueh B R, et al. “Treatment of Bepharospasm with Botulinum Toxin. A preliminary report” Arch Ophthalmol 102 (1984) 1464. Antibody production is thought to be a function of cumulative dose, antigenic load per dose, and time interval between injections. Thus, it has been suggested that the smallest therapeutic dose be given with maximum time interval between sequential injections. Given the importance of the use of BoNT in treating certain diseases, use of BoNT-F is under investigation in patients who have become immunologically resistant to serotypes A and B.
Although the botulinum toxins are effective for the majority of patients, therapeutic effect can vary widely independent of detectable antibody formation—not only from individual to individual, but also for an individual from treatment to treatment. Further, some populations have been identified to be more likely to show poor- or non-responsiveness to the toxins. For example, up to 30% of patients over the age of 65 may demonstrate decreased botulinum toxin efficacy. The basis of failure of a patient to respond to treatment can be difficult to discern, given that possible explanations include original manufacturer differences in potency, the possibility that the patient has developed toxin specific antibodies, as well as the possibility that the product has degraded or been agitated during reconstitution. However, diminished effect within a particular patient population suggests the possibility of another factor as well.
Reports of patient demise from the putative dose-dependent distant spread of very large amounts of botulinum toxin administration have surfaced in the last few years, so any methods which may decrease the dose of toxin necessary to achieve a desired therapeutic outcome would be beneficial.
From the foregoing, it is apparent that any ability to assure maximal patient responsiveness in treatment and to provide closer standardization of potency would represent an important therapeutic and safety advance.